The synthesis and purification of single stranded oligonucleotides is of great importance to molecular biologists due to their uses as probes and their uses in recombinant DNA technology. The oligomers of greatest interest range in size from about 15 to 50 bases in length. With the improvements in the technology of oligonucleotide synthesis over the past few years, the need for rapid separations and purification of the synthetic oligonucleotides is becoming paramount.
Oligonucleotides were first separated by anion exchange chromatography on alkylammonium moieties bonded to polysaccharide based packing media as described by H. R. Matthews [Eur. J. Biochem., Vol. 7, 96 (1968)]. These methods had the disadvantage of being limited to relatively short chain oligonucleotides, having extremely long analysis times, and poor resolution of the individual oligomers. The large particle sizes used for the packing material contributed to these difficulties.
T. F. Gabriel and J. E. Michalewsky, [J. Chromatogr., Vol. 80, 263 (1973)] described the use of a pellicular ion exchange packing for the high performance ion exchange separation of oligonucleotides. This material allowed the separation in a shorter time, but only short chain oligonucleotides were investigated. M. J. Gait and R. C. Sheppard, [Nucleic Acid Res., Vol. 4, 1135 (1977)] reported the use of a microparticulate silica based strong anion exchanger which allowed the separation of deoxythymidyl oligomers up to seven bases in length within 20 minutes. This method still suffers from the inability to resolve longer oligonucleotides.
W. Haupt and A. Pingoud [J. Chromatogra., Vol. 260, 419 (1983)] used the same packing with a modified mobile phase to separate oligomers of deoxyadenosine and obtained a degree of resolution to oligomers of around 25 to 30 bases in length. The actual size of the individual oligonucleotides was poorly defined and baseline resolution was not achieved for oligomers more than approximately 15 bases long. These authors reported that the separations achieved by ion exchange chromatography were dependent upon the chain length of the oligonucleotide. No evidence of purity of the isolated materials was given.
The anion exchange chromatography of oligonucleotides on a polymer based strong anion exchange material was reported by M. V. Cubellis, et al. [J. Chromatogr., Vol. 329, 406 (1985)] for the separation of 15 to 18 base long oligonucleotides within 50 to 60 minutes. They report that at alkaline pH the separations became less influenced by the base sequency of the oligonucleotide.
W. Jost, et al. [J. Chromatogr. Vol. 185, p. 403 (1979)] reported the synthesis of a weak anion exchanger prepared from the reaction of N,N-dimethyl-2-hydroxyethylamine with glycidoxypropyl-substituted silica. They reported that the separation of oligonucleotides was related to their secondary structure. The isocratic separation of oligouridylic acids of up to 9 bases in length within 20 minutes was reported. This support still does not allow separation of longer chain oligonucleotides.
The use of an aminopropyl-silica weak anion exchanger for the separation of small heterooligonucleotides was reported by L. W. McLaughlin, et al. [Anal. Biochem., Vol. 112, 60 (1981)]. Retention was shown to be dependent on chain length and on the sequence and nature of the bases comprising the oligonucleotide. Oligonucleotides up to only 12 bases in length were separated in a time of 100 minutes.
Polyethyleneimine, crosslinked on silica gel, was used by J. D. Pearson and F. E. Regnier, [J. Chromatogr., Vol. 255, 137 (1983)] as a weak anion exchange material for the separation of oligoadenylic acids and oligothymidylic acids. Resolution of oligo(A) up to 17 bases and oligo(T) up to 15 bases was achieved in an isocratic mode in 80 and 60 minutes, respectively. This use of a slowly changing gradient profile allowed the separation of oligo(A) as high as 35-mer, albeit over 5 hours and with poorer resolution. No separations of hetero-oligomers were demonstrated.
Methylation of polyethyleneimine, crosslinked on silica (3 .mu.m diameter) was reported by R. R. Drager and F. E. Regnier [Anal. Biochem., Vol. 145, 47 (1985)] to improve the separation of oligonucleotides. A gradient separation of oligo(A) in the 40 to 60 base range was reported. They also reported the chromatography of oligo(A) of up to 18 bases and oligo(T) up to 24 bases. In addition, the chromatography of two hetero-oligomers of 18 bases in length were reported. No independent data (such as gel electrophoresis) was given to assess the purity of the isolated components. The chromatography required 2 hours to achieve separation of the hetero-oligomers.
Mixed-mode chromatographic separations of multifunctional compounds have been known for some time. J. Visser and M. Strating [Biochem. Biophys. Acta., Vol. 384, 69 (1975)] reported the mixed mode separation of proteins on a stationary phase prepared from the reaction of aminohexanol with cyanogen bromide activiated Sepharose. This phase was noted to show results very similar to those from a similar phase prepared from propylamine. No separations of oligonucleotides was reported.
G. C. Walker, et al. [Proc. Nat. Acad. Sci. USA, Vol. 72, 122 (1975)] described the use of a quaternary ammonium salt coated on a Kel-F particle (designated as RPC-5) as a packing material for the separation of oligonucleotides. Typical operating conditions for this material were reported by R. D. Wells, et al. [Meth. Enzymol., Vol. 65, 327 (1980)]. These workers demonstrated the separation of oligodeoxyadenylic acid up to 40 base units long within 3 hours. The major disadvantage with this material was bleed of the quaternary ammonium phase, especially at low ionic strength.
Packings designed to exploit mixed mode separations for oligonucleotides were developed by R. Bischoff and L. W. McLauchlin [J. Chromatogr., Vol. 270, 117 (1983)]. They reacted organic aminoacids with aminopropyl silica to make phases with both weak anion exchange and reversed phase properties. These workers studied only oligomers up to 10 bases in length, the separation of which took 60 mintutes. This support allowed separation of oligonucleotides of the same chain length based upon base sequence.
J. B. Crowther, et al. [J. Chromatogr., Vol. 282, 619 (1983)] described an alternative approach in which they bonded two different silanes to silica, one a reversed phase C8, the other a chloropropylsilane which was subsequently converted to a strong anion exchange group by reaction with dimethylbenzylamine. This material allowed rapid separations, a 13 base oligouridylic acid was analyzed in 20 minutes, and a hetero-oligonucleotide 15 units long in 29 minutes. Again, the separation of long oligonucleotides was not reported.
In an attempt to mimic the RPC-5 packing (which is no longer commercially available), R. Bischoff and L. McLauchlin [Anal. Biochem., Vol. 151, 526 (1985)] coated on octadecylsilica with methyloctylammonium chloride. The separation of oligomers of deoxyuridylic acid up to 90 units in length over a period of 19 hours was shown, although baseline resolution was lost beyond components greater than 30 bases long which eluted in 12 hours. The main purpose of this work was to purify tRNA rather than the synthetic oligonucleotides. The major disadvantages of the material are the long analysis times and the potential of column bleed.
Other techniques of liquid chromatography have also been used for the purification of oligonucleotides. Reversed phase chromatography of both protected and unprotected oligonucleotides up to 12 base units in length was reported by H-J Fritz, et al. [Biochemistry, Vol. 17, 1257 (1978)]. No apparent correlation between chain length of oligoncleotide and retention was observed.
A. M. Delort, et al., [J. Chromatogr., Vol. 283, 462 (1984)] reported the separation of a variety of hetero-oligonucleotides up to 19 bases long by reversed phase chromatography. These analyses were rapid, being complete in less than 20 minutes but no correlation of retention time with chain length was seen.
Reversed phase ion-pair chromatography has been used for the separation of oligonucleotides. W. Haupt and A. Pingaud [J. Chromatogr., Vol. 260, p. 419 (1983)] have reported a gradient method for the analysis of oligonucleotides up to 16 bases long. The separation was related to chain length and was accomplished in about 45 minutes. The disadvantage of ion pair methods is that the ion pair reagent must be removed from the collected fractions. In addition, the resolution between homologues was shown to be less in ion pair than with ion exchange chromatography as larger oligonucleotides are separated.
Charge transfer chromatography of oligonucleotides using acriflavin bonded to agarose has been described by J. M. Egly [J. Chromatogr., Vol. 215, 243 (1981)]. This was used only for short chain compounds (up to 8-mer) and was very slow, typical separation times being on the order of 6 hours.
The enhancement of stability of bonded phase packings and silica to hydrolysis by buffers at pH values above 7 by introducing zirconium atoms into the silica structure has been disclosed by Stout (U.S. Pat. No. 4,600,646 issued July 15, 1986). Packings for the size exclusion chromatography of biopolymers were shown to have greater stability than similar materials which has not been so stabilized. No separations of oligonucleotides was reported.
It is thus apparent that a number of methods and materials are known for the separation of oligonucleotides, none of which meet all of the desirable criteria for separation and purification of oligonucleotides. Those desirable criteria are: rapid separation (generally less than 1 hour); separation based upon chain length resolution sufficient to allow recovery of substantially pure oligonucleotides; and being sufficiently stable to allow reproducible separations and preventing contamination of the product with released bonded phase. Most known separations are limited to small (up to 20 bases) oligonucleotides and require 1 to 2 hours.
The methods and materials which allow separation of longer oligonucleotides generally require very much longer analysis times and typically do not give good resolution between homologues. The need remains for a rapid separation of oligonucleotides in the 10 to 45-mer range which allows isolation of individual oligomers with high purity.